High fat diet induces early increase of CTCs detectable with intravital flow cytometry. (A) A dual-color M109 cell line which expresses both GFP and RFP. M109 cells in a tissue culture (upper panels) and in a primary tumour tissue (lower panels). (B) Intravital flow cytometry using a superficial vein of a mouse ear by dual-color confocal detection of CTCs. CARS: grey, transmission: light blue. CTCs were positively indentified based on both GFP (green) and RFP (red) signals generated by one-photon dual-laser excitations. Rightmost two panels were processed with ImageJ software to remove uncorrelated background noise. (C) High fat diet induced early increase of CTCs in the blood stream. Solid circles and squares represent average CTCs/minute measured in each HD and LD mouse, respectively. Error bars represent distribution of CTC/minute across four repeated measurements in each mouse. Open circles and squares represent average CTCs/minute of 8 HD and LD mice, respectively. No CTC detected on week 1 in mice of either diet group. (D) Isolated lipid-rich CTCs from LD (upper panels) and HD (lower panels) mice identified with CARS imaging for lipid and TPEF imaging for GFP (green) and RFP (red) expression.